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In the retina, cell fate determination is thought to be regulated by a series of local cell-cell interactions. Evidence suggests that retinal precursors utilize Notch-mediated intercellular signaling to regulate their fates. However, the identity of the endogenous ligand and its role in the Notch-signaling pathway is not well understood. We have identified C-Delta-1 as the putative endogenous ligand for Notch, in the developing chick retina. C-Delta-1 is coexpressed spatially and temporally with C-Notch-1 and their expression is associated with the temporal aspects of cell birth in the developing retina. This suggests that Delta-Notch signaling is utilized to maintain progenitors in an uncommitted state and that a subtle fluctuation in this signaling helps to sort out competent cells during successive cell-fate determination. We have tested the latter possibility in the specification of the ganglion cells. In early stages of retinal development when ganglion cells are the predominant cells born, decreasing C-Delta-1 expression with antisense oligonucleotides increases the proportion of RA4 antigen-expressing ganglion cells which are recruited predominantly in the periphery. Conversely, use of exogenous Drosophila Delta leads to a decrease in the RA4 antigen-expressing ganglion cells. Our results suggest that C-Delta-1 activation of the Notch pathway regulates the specification of retinal neurons in general and of ganglion cells in particular.

NeuroD, a vertebrate homolog of Drosophila atonal gene, plays an important role in the differentiation of neuronal precursors (Lee et al., 1995). We have investigated whether NeuroD subserves a similar function in mammalian retinal neurogenesis. Expression of NeuroD is detected in successive stages of retinal neurogenesis and is associated with a differentiating population of retinal cells. The association of NeuroD predominantly with postmitotic precursors in early as well as late neurogenesis suggests that NeuroD expression plays an important role in the terminal differentiation of retinal neurons. The notion is supported by observations that overexpression of NeuroD during late neurogenesis promotes premature differentiation of late-born neurons, rod photoreceptors, and bipolar cells, and that NeuroD can interact specifically with the E-box element in the proximal promoter of the phenotype-specific gene, opsin.

Cell fate specification is regulated in part by lateral inhibition mediated by Notch signaling. Notch signaling is negatively regulated by Numb, an intrinsic factor that regulates cellular competence. In this study we have examined the involvement of Numb in retinal development, which has been shown to be influenced by Notch signaling. In the developing retina, Numb is asymmetrically distributed towards the ventricular and vitreal poles of different cells. Asymmetric localization is evident not only in mitotic cells but in postmitotic ganglion cells as well, suggesting that the subcellular distribution of Numb may play a role after cells have exited the cell cycle. This is supported by the expression of Numb in terminally differentiated neurons in the adult retina. Although Numb is an intrinsic factor, it is observed that its subcellular distribution is influenced by epigenetic cues such that a higher proportion of cells cultured at high density express Numb asymmetrically. A correlation is observed between asymmetric localization and cellular competence; cells in which Numb is asymmetric differentiate more readily in culture than those that express Numb symmetrically. We have identified alternative splice variants in the developing and adult retina that correspond to isoforms that have been shown to regulate proliferation and differentiation. The dynamic temporal expression patterns of alternative splice variants and isoforms suggest that Numb may influence proliferation and differentiation of retinal progenitors during neurogenesis and maturation of postmitotic neurons. Together, these results demonstrate the complex role of the distribution of Numb within progenitors and postmitotic neurons.

It is believed that signaling through the epidermal growth factor (EGF) receptor plays a critical role in the development of Drosophila eyes. In the present study we have analyzed the role that EGF-mediated signaling plays in vertebrate retinal development. We have observed that during late retinal neurogenesis EGF delays rod photoreceptor differentiation and that this effect of EGF involves the modulation of expression of a homologue of Drosophila proneural genes, Mash1. EGF causes a significant decrease in Mash1 expression and an increase in the proportion of proliferating cells in the retina in vitro. The decrease in Mash1 expression is accompanied by a concomitant decrease in opsin expression, a marker for overt rod photoreceptor differentiation. Withdrawal of EGF leads to an increase in both Mash1 and opsin expression; however, the onset of expression of Mash1 precedes that of opsin. Our study identifies a proliferative intermediate precursor, characterized by Mash1 expression, that is the target of EGF-mediated suppression of rod photoreceptor differentiation. Based on the evolutionarily conserved roles of EGF- and Notch-mediated signaling in the delay of differentiation in proliferating precursors we propose that these distinct signaling mechanisms act in concert to ensure the fidelity of the strict temporal and spatial nature of cell fate determination in the retina.

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